Guinea Pig Horizontal Cells Express GABA the GABA-Synthesizing Enzyme GAD65 and the GABA Vesicular Transporter

摘要

γ-Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD67, GAT-1, or GAT-3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells.